FIGURE 5.

SecG prevents dislocation of inserted SecA. A and B, proOmpA translocation into 4 m urea-washed IMV prepared from K003 (SecG⁺ IMV) or KN553 (ΔSecG IMV) was carried out at 37 °C using either ¹²⁵I WT SecA (A) or ¹²⁵I D209N SecA (B) as described under “Experimental Procedures.” At 20 min, cold WT SecA (60 μg/ml) was added to the reactions. An aliquot (100 μl) was withdrawn at each indicated time and chilled on ice, followed by proteinase K digestion (1 mg/ml). The membrane-protected fragment of ¹²⁵I-labeled SecA of 30 kDa was quantitated and plotted against reaction time. C, ProOmpA translocation was initiated as described in B using ¹²⁵I D209N SecA (0.4 μg/ml). At 20 min, cold WT SecA was added at the indicated concentrations, and the reaction was continued for a further 20 min. Samples were then chilled and digested with proteinase K (1 mg/ml). The membrane-protected fragment of ¹²⁵I-labeled SecA of 30 kDa was quantitated. It was expressed as a percentage of that in the respective IMV at 0.1 μg/ml and plotted against SecA concentration.